Table 1.
Identification and classification of Tc-SMP genes from T. cruzi genomes deposited in TriTrypDB and GenBank.
Table 2.
Tc-SMP proteins secreted into the extracellular medium by epimastigotes and metacyclic trypomastigotes (clone Dm28c) identified by proteomic analysis.
Fig 1.
Alignment of TcSMP amino acid sequences of T. cruzi—clone CL Brener (CLB).
Sequences TcCLB.510129.20, TcCLB.510129.30, TcCLB.509639.10, TcCLB.507711.90, extended TcCLB.507711.100, extended TcCLB.507711.110 and TcCLB.508173.120 were deposited in the GenBank database by the T. cruzi Genome Project. Clones 23B, 24B and 24C (GenBank KJ682657, KJ682658 and KJ682659) were isolated in this work from genomic DNA (CLB) by PCR. Sequences were aligned by the MegAlign program (DNASTAR). Black, gray and light gray blocks indicate residues with 100%, 80% and 60% identity, respectively. Colored lines above the sequences denote the predicted signal peptide in green, transmembrane domains in orange and uncleaved anchor signal in blue. Red arrows indicate the putative initiator methionine. Yellow boxes correspond to peptides found in the proteomic analysis of T. cruzi membrane protein-enriched fractions [27]. Red boxes correspond to peptides identified in the T. cruzi secretome [43]. Blue boxes correspond to the YGQ motif present in Procyclic Surface Specific Antigen-2 (PSSA-2) of T. brucei.
Fig 2.
Modular architecture of TcSMP paralogs and orthologs.
Left Panel) Schematic representation of TcSMP proteins from T. cruzi isolates (CLB, Sylvio X10/1 and Dm28c) and T. cruzi marinkellei. Right Panel) Schematic representation of TcSMP orthologs in T. brucei, T. brucei gambiense, T. congolense, T. vivax, T. rangeli and T. grayi. The amino acid sequence is represented by a black bar and the first and last residues are indicated below. The red vertical lines indicate the putative in frame initiator methionine residues. The positions of predicted signal peptide (SP), signal anchor (SA) and transmembrane domain (TM) are indicated by green, blue and orange lines, respectively. Sequences were identified by their entries in TriTrypDB, with the exception of those from T. rangeli and T. grayi, which were identified by GenBank accession numbers. T. cruzi sequences: TcCLB, clone CL Brener; TCSYLVIO, clone Sylvio X10/1; TCDM, clone Dm28c; Tc_MARK, T. cruzi marinkellei.
Fig 3.
Western blotting analysis of TcSMP proteins in the different developmental stages of T. cruzi and in the procyclic form of T. brucei rhodesiense.
A) Samples of T. cruzi (CL strain) (1 x 107 cells/slot) and T. brucei (5 x 106 cells/slot) were lysed in Laemmli sample buffer. Proteins were separated by electrophoresis on SDS- polyacrylamide gel (10%), transferred to a nitrocellulose membrane and reacted against anti-TcSMP polyclonal antibodies. Antibody against a constitutively expressed protein (tubulin) was used as a loading control. T. cruzi developmental forms: epimastigotes (E), metacyclic trypomastigotes (M), extracellular amastigotes (A) and tissue culture trypomastigotes (T). T. brucei rhodesiense procyclic forms. B) Two μg of the recombinant TcSMP-GST purified protein was separated by SDS-PAGE and reacted against anti-TcSMP and anti-GST antibodies. The recombinant protein is coded by a 311-bp fragment of TcSMP in phase with GST (see methods and S4 Fig). Molecular mass markers in kilodaltons (kDa) are indicated on the left and reactive proteins molecular mass on the right.
Fig 4.
Cellular distribution of TcSMP proteins in different stages of the T. cruzi life cycle.
Indirect immunofluorescence with anti-TcSMP antibodies in permeabilized (left) or non-permeabilized (right) T. cruzi (CL strain). T. cruzi developmental forms: epimastigotes (E), metacyclic trypomastigotes (M), extracellular amastigotes (A) and tissue culture trypomastigotes (T). Labeling with DAPI and TcSMP proteins is shown in blue and green, respectively. Bar: 10 μm.
Fig 5.
Recombinant protein TcSMP properties in host cell binding, Ca2+ signaling, lysosome scattering and inhibition of T. cruzi metacyclic trypomastigote invasion.
A) HeLa cells were incubated with the indicated proteins, at varying concentrations, and binding was evaluated as described in the methods section. Values are the means ± SD of triplicates of one representative assay out of three. B) HeLa cells were grown overnight in DMEM with 10% serum on ibidi multichamber dishes (Hi-Q4, ibidi), and the fluorescence intensity of Fluo-4 was determined as described in the methods section. Images show the basal fluorescence at time zero (left panel) and the maximum fluorescence after stimulation with 40 μg/mL purified TcSMP or GST (right panel). Note the increase in the fluorescence intensity after challenge with TcSMP but not with GST. Bar: 50 μm. C) HeLa cells were grown as in (B) and intracellular calcium was quantified after stimulation (black arrow) with recombinant protein TcSMP or GST. The graph shows the relative concentration of cytoplasmic Ca2+ at 400 sec after stimulation, expressed as the maximum peak of total fluorescence minus basal fluorescence at time zero. Thirty cells in three independent experiments were analyzed. Shown on the left side is the difference between the maximum calcium concentration at 400 sec after stimulation with TcSMP and GST (*p = 0.0005). D) HeLa cells were incubated for 30 min with 40 μg/mL of TcSMP or GST, and then processed for confocal fluorescence analysis using anti-LAMP2 antibody and Alexa Fluor 488-conjugated anti-mouse IgG (green), phalloidin-TRITC (red) for actin visualization and DAPI (blue) for DNA, under 63X objective. (Bar: 20 μm). Note the lysosome mobilization to the host cell periphery after treatment with TcSMP. E) HeLa cells were incubated with the indicated recombinant protein at 40 μg/mL. After 30 min, CL strain metacyclic trypomastigotes were added and incubation proceeded for 1 h before fixation and staining with Giemsa. The number of internalized parasites was counted in a total of 250 cells. Values are the means ± SD of three independent assays performed in duplicate. TcSMP, but not GST, significantly inhibited parasite invasion (*p < 0.05).
Fig 6.
Comparative analysis of lysosome scattering and T. cruzi cell invasion inhibitory effects of TcSMP and gp82 proteins.
A) HeLa cells were incubated for 30 min with TcSMP or gp82, at the indicated concentrations then processed for confocal fluorescence analysis using anti-LAMP2 antibody and Alexa Fluor 488-conjugated anti-mouse IgG (green), and DAPI (blue) for DNA, under 63X objective. (Bar: 10 μm). Note the lysosome mobilization to the host cell periphery after treatment with 40 μg/mL TcSMP. B) HeLa cells were incubated with CL strain metacyclic trypomastigotes in absence or in the presence of TcSMP, gp82 or the combination of the two proteins, at the indicated concentrations. After 1 h incubation, the cells were fixed and stained with Giemsa. The number of internalized parasites was counted in a total of 250 cells. Values are the means ± SD of three independent assays performed in duplicate. Inhibition by TcSMP at 40 μg/mL, by gp82 at 20 μg/mL or 40 μg/mL and by combined proteins, was significant (*p < 0.05).
Fig 7.
Genomic organization of the TcSMP family.
A) Schematic representation of in silico chromosomes TcChr37-P, TcChr37-S and TcChr27-P showing the distribution of TcSMP genes (red boxes). The accession numbers of TcSMP genes in TriTrypDB are indicated above. B) Restriction Southern blot analysis of TcSMP loci. Genomic DNA of CLB was digested with different restriction enzymes, separated on a 0.8% agarose gel and transferred to nylon membrane. Autoradiogram obtained by hybridization with (32P) TcSMP probe. The enzymes are indicated above each lane. The colored boxes indicate the restriction fragments predicted in chromosomes TcChr37-P, TcChr37-S, and TcChr27-P. C) Karyotype mapping of TcSMP genes. Chromosomal bands of CLB, strain G and T. cruzi marinkellei were separated by PFGE, stained with ethidium bromide, transferred onto nylon membranes and hybridized with TcSMP probe. Numbers on the left correspond to the sizes (Mb) of Hansenula wingei chromosomes used as markers, and on the right the sizes of chromosomal bands hybridizing with (32P) TcSMP probe.
Fig 8.
Synteny of TcSMP genes among T. brucei, T. cruzi and T. grayi.
Genomic regions around the T. cruzi (CLB) TcSMP paralogs and T. brucei and T. grayi orthologs are shown. Analyses were conducted via TBLASTN using the Artemis Comparison Tool (ACT) [37] with an E value of 500. Comparison among TcChr37 (‘‘P” chromosome assigned to the non-Esmeraldo haplotype and ‘‘S” to the Esmeraldo haplotype), T. brucei chromosome Tb10 and the contig Tgr_12_V1 of T. grayi. Homologous genes are connected by colored lines. The matches and reverse matches are represented in red and in blue, respectively. Grey blocks represent each chromosome. Chromosome markers are drawn in the sense (+) and antisense (-) strands. The numbers indicate the location on chromosomes: T. brucei TREU927 (CHR10—position: 2729733–2761348 nt), CLB T. cruzi (TcChr37-S—positions: 848335–873376 nt and TcChr37-P 848287–882361 nt) and contig of T. grayi (Tgr_12_V1 –position: 1–64810 nt). The locations of the TcSMP paralogs and T. brucei and T. grayi orthologs are depicted in chromosomes by red blocks. Abbreviations: TcSMP (TcSMP—T. cruzi/ PSSA-2—T. brucei); HIRA (HIRA-interacting protein 5, putative); ubiqui (ubiquitin-like modifier-activating enzyme ATG7, putative); RNA-bp (RNA-binding protein, putative); ubiqui E1 (ubiquitin activating E1 enzyme, putative); surf glyco (surface glycoprotein, putative); PRC (paraflagellar rod component, putative); HP (hypothetical protein, conserved); S-methyl (S-methyl-5thioribose kinase); POT (proton-dependent oligopeptide transporter, POT family); tetratrico (tetratricopeptide domain 4); 60S (putative 60S ribosomal protein L6); sterol C (sterol C-24 reductase); IFT 57 (predicted: intraflagellar transporter protein 57 homolog); tRNA-s lig (tRNA-splicing ligase RtcB); PP 2A (protein phosphatase 2A regulatory subunit).
Fig 9.
Phylogenetic tree of TcSMP sequences built by the Maximum-Likelihood method.
The phylogram was constructed from the alignment of amino acid sequences of TcSMP proteins and their homologs in other trypanosomes. The ML tree was constructed using Phylip. Red and blue lines on the right delimit the American and African trypanosome clusters, respectively. Genes located on chromosome platforms TcChr37-P, TcChr37-S and TcChr27-P are highlighted in green, violet, and pink, respectively. Sequences were identified by their entries in TriTrypDB, except those from T. rangeli (AGN32959, ESL09843 and ESL05362) and T. grayi (KEG05859), which were identified by their GenBank accession numbers. TriTrypDB entries are prefixed by species and strain followed by the number of the sequence. T. cruzi: TcCLB., clone CL Brener; TCDM_, clone Dm28c; TCSylvio_, clone Sylvio X10/1; T. cruzi marinkellei: Tc_MARK_; T. brucei brucei: Tb927., strain TREU; Tb427., strain Lister; T. vivax: TvY486_; T. congolense: TcIL3000. Clones 23B, 24B and 24D (GenBank KJ682657, KJ682658 and KJ682659) were isolated in this work by PCR amplification using TcSMP specific primers. A trans-sialidase from T. cruzi CLB (XP_816001) was used as the outgroup.